Analogs of parathyroid hormone and parathyroid hormone related peptide: synthesis and use for the treatment of osteoporosis

ABSTRACT

Synthetic polypeptide analogs of parathyroid hormone PTH, parathyroid hormone related peptide PTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, in which amino acid residues (22-31) form an amphipathic α-helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence: 
     
         Haa(Laa Laa Haa Haa).sub.2 Laa 
    
     and their pharmaceutically acceptable salts are useful for the prophylaxis and treatment of ostoporosis in mammals. Processes for the production of the polypeptides via solid phase and recombinant methods are provided.

BACKGROUND OF THE INVENTION

a) Field of the Invention

This invention relates to novel analogs of parathyroid hormone andparathyroid hormone related peptide, their synthesis by solid phase andrecombinant techniques, and their use for increasing bone mass inmammalian subjects.

b) Description of Related Art

Osteoporosis is the most common form of metabolic bone disease and maybe considered the symptomatic, fracture stage of bone loss (osteopenia).Although osteoporosis may occur secondary to a number of underlyingdiseases, 90% of all cases appear to be idiopathic. Postmenopausal womenare particularly at risk for idiopathic osteoporosis (postmenopausal orType I osteoporosis). Another high risk group for idiopathicosteoporosis is the elderly of either sex (senile or Type IIosteoporosis). Osteoporosis has also been related to corticosteroid use,immobilization or extended bed rest, alcoholism, diabetes, gonadotoxicchemotherapy, hyperprolactinemia, anorexia nervosa, primary andsecondary amenorrhea, and oophorectomy.

In the various forms of osteoporosis, bone fractures, which are theresult of bone loss that has reached the point of mechanical failure,frequently occur. Postmenopausal osteoporosis is characterized byfractures of the wrist and spine, while femoral neck fractures seem tobe the dominant feature of senile osteoporosis.

The mechanism by which bone is lost in osteoporotics is believed toinvolve an imbalance in the process by which the skeleton renews itself.This process has been termed bone remodeling. It occurs in a series ofdiscrete pockets of activity. These pockets appear spontaneously withinthe bone matrix on a given bone surface as a site of bone resorption.Osteoclasts (bone dissolving or resorbing cells) are responsible for theresorption of a portion of bone of generally constant dimension. Thisresorption process is followed by the appearance of osteoblasts (boneforming cells) which then refill with new bone the cavity left by theosteoclasts.

In a healthy adult subject, the rate at which osteoclasts andosteoblasts are formed is such that bone formation and bone resorptionare in balance. However, in osteoporotics an imbalance in the boneremodeling process develops which results in bone being lost at a ratefaster than it is being made. Although this imbalance occurs to someextent in most individuals as they age, it is much more severe andoccurs at a younger age in postmenopausal osteoporotics or followingoophorectomy.

There have been many attempts to treat osteoporosis with the goal ofeither slowing further bone loss or, more desirably, producing a netgain in bone mass. Certain agents, such as estrogen and thebisphosphonates, appear to slow further bone loss in osteoporotics.Agents which slow bone loss, because of the different durations of boneresorption and formation, may appear to increase bone mass (on the orderof 3 to 7%). However, this apparent increase is limited in time, notprogressive, and is due to a decrease in "remodeling space." Inaddition, because of the close coupling between resorption andformation, treatments which impede bone resorption also ultimatelyimpede bone formation.

It has been suggested that treatment with parathyroid hormone (PTH)would lead to both increased bone turnover and a positive calciumbalance. However, human clinical trials have shown that any increase intrabecular bone is offset by a decrease in cortical bone, so that thereis no net increase in total bone.

Hefti, et al. in Clinical Science 62, 389-396 (1982) have reported thatdaily subcutaneous doses of either bPTH(1-84) or hPTH(1-34) increasedwhole body calcium and ash weight of individual bones in both normal andosteoporotic adult female rats.

Liu, et al. in J. Bone Miner. Res. 6: 10, 1071-1080 (1991) have notedthat ovariectomy of adult female rats induced a 47% loss in thepercentage of trabecular bone in the proximal tibial metaphysis,accompanied by a significant increase in the number of osteoblasts andtrabecular osteoclasts. Daily subcutaneous injections of hPTH(1-34)completely reversed the loss of trabecular bone and resulted in amountsof trabecular bone exceeding that of sham operated controls. The numberof osteoblasts increased and the number of osteoclasts decreased.

Hock et al. in J. Bone Min. Res. 7: 1, 65-71 (1992) have reported thatdaily subcutaneous injections of hPTH(1-34) to healthy adult male ratsfor 12 days increased trabecular and cortical bone calcium and dryweight. Total bone mass, trabecular bone volume, trabecular thicknessand number, and osteoblastic surfaces were increased.

The mammalian parathyroid hormones, e.g. human (hPTH), bovine (bPTH),and porcine (pPTH), are single polypeptide chains of 84 amino acidresidues, with molecular weights of approximately 9500. Biologicalactivity is associated with the N-terminal portion, with residues (1-34)apparently the minimum required.

The N-terminal segment of human PTH differs from the N-terminal segmentof the bovine and porcine hormones by only three and two amino acidresidues, respectively: ##STR1##

The primary function of PTH is to elicit the adaptive changes that serveto maintain a constant concentration of Ca²⁺ in the extracellular fluid.PTH acts on the kidneys to increase tubular reabsorption of Ca²⁺ fromthe urine, as well as stimulating the conversion of calcifediol tocalcitriol, which is responsible for absorption of Ca²⁺ from theintestines. One prominent effect is to promote the mobilization of Ca²⁺from bone. PTH acts on bone to increase the rate of resorption of Ca²⁺and phosphate. PTH stimulates the rate of bone resorption byosteoclasts, increases the rate of differentiation of mesenchymal cellsto osteoclasts, and prolongs the half life of these latter cells. Withprolonged action of PTH the number of bone forming osteoblasts is alsoincreased; thus, the rate of bone turnover and remodeling is enhanced.However, individual osteoblasts appear to be less active than normal.

Rosenblatt, et al. in U.S. Pat. Nos. 4,423,037, 4,968,669 and 5,001,223have disclosed PTH antagonists obtained by the deletion of theN-terminal (1-6) amino acids and the selective replacement of Phe⁷,Met⁸,18, and Gly¹². Tyr³⁴ -NH₂ reportedly increased the activity andstability of these compounds.

Parathyroid hormone-related peptide (PTHrp), a 140+ amino acid protein,and fragments thereof, reproduce the major biological actions of PTH.PTHrp is elaborated by a number of human and animal tumors and othertissues and may play a role in hypercalcemia of malignancy. The sequenceof hPTHrp (1-34) is as follows: ##STR2##

The sequence homology between hPTH and hPTHrp is largely limited to the13 N-terminal residues, 8 of which are identical; only 1 of 10 aminoacids in the (25-34) receptor binding region of hPTH is conserved inhPTHrp. Conformational similarity may underlie the common activity.Cohen, et al. in J. Biol. Chem. 266: 3, 1997-2004 (1991) have suggestedthat much of the sequence of PTH(1-34) and PTHrp(1-34), in particularregions (5-18) and (21-34), assumes an α-helical configuration, whilenoting that there is some question whether this configuration prevailsfor the carboxyl terminal end under physiological conditions. Such asecondary structure may be important for lipid interaction, receptorinteraction, and/or structural stabilization.

We have synthesized analogs of PTH and of PTHrp with the objective ofdeveloping improved therapeutic agents for the restoration of bone massin mammalian subjects, including those afflicted with osteoporosis.

SUMMARY OF THE INVENTION

This invention provides synthetic polypeptide analogs of parathyroidhormone (PTH), parathyroid hormone related peptide (PTHrp), and of thephysiologically active truncated homologs and analogs of PTH and PTHrp,and salts thereof, in which amino acid residues (22-31) form anamphipathic α-helix, said residues (22-31) selected from hydrophilicamino acids (Haa) and lipophilic amino acids (Laa) ordered in thesequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

When specific illustrative embodiments of this sequence type areinserted into PTH, PTHrp, and the physiologically active truncatedanalogs and homologs of PTH and PTHrp, the resulting polypeptides areeffective bone remodeling agents.

In one aspect, then, this invention provides analogs of PTH, PTHrp, andof the physiologically active truncated homologs and analogs of PTH andPTHrp, or salts thereof, in which amino acid residues (22-31) form anamphipathic α-helix, the sequence of said residues (22-31) selectedfrom: ##STR3##

Also provided are pharmaceutical compositions for the prevention ortreatment of conditions characterized by decreases in bone masscomprising an effective bone mass increasing amount of a polypeptideanalog of parathyroid hormone (PTH), parathyroid hormone related peptide(PTHrp), and of the physiologically active truncated homologs andanalogs of PTH and PTHrp, and salts thereof, in which amino acidresidues (22-31) form an amphipathic α-helix, said residues (22-31)selected from hydrophilic amino acids (Haa) and lipophilic amino acids(Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

Further, this invention provides methods for treating mammalianconditions characterized by decreases in bone mass which methodscomprise administering to a mammal in need thereof an effective bonemass increasing amount of a polypeptide analog of PTH, PTHrp, or of aphysiologically active truncated homolog or analog of PTH or PTHrp, or asalt thereof, in which amino acid residues (22-31) form an amphipathicα-helix, said residues (22-31) selected from hydrophilic amino acids(Haa) and lipophilic amino acids (Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

More specifically, this invention provides methods for treatingmammalian conditions characterized by decreases in bone mass whichmethods comprise administering to a mammal in need thereof an effectivebone mass increasing amount of a polypeptide analog of PTH, PTHrp, or ofa physiologically active truncated homolog or analog of PTH or PTHrp, orsalt thereof, in which amino acid residues (22-31) form an amphipathicα-helix, the sequence of said residues (22-31) selected from: (SEQ IDNOS: 26, 27, 28, 29, and 30).

This invention also includes processes for the solid phase synthesis ofpolypeptide analogs of PTH, PTHrp, and of the physiologically activetruncated homologs and analogs of PTH and PTHrp, and salts thereof, inwhich amino acid residues (22-31) form an amphipathic α-helix, saidresidues (22-31) selected from hydrophilic amino acids (Haa) andlipophilic amino acids (Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa,

which processes comprise sequentially coupling protected amino acids ona suitable resin support, removing the side chain and N.sup.α-protecting groups, and cleaving the polypeptide from the resin.

This invention also encompasses processes for the solid phase synthesisof polypeptide analogs of PTH, pTHrp, and of the physiologically activetruncated homologs and analogs of PTH and PTHrp, and salts thereof, inwhich amino acid residues (22-31) form an amphipathic α-helix, thesequence of said residues (22-31) selected from: (SEQ ID NOS: 26, 27,28, 29, and 30); which processes comprise sequentially couplingprotected amino acids on a suitable resin support, removing the sidechain and N.sup.α -protecting groups, and cleaving the polypeptide fromthe resin.

Also included are DNA sequences, vectors, and plasmids for therecombinant synthesis of polypeptide analogs of PTH, PTHrp, and of thephysiologically active truncated homologs and analogs of PTH and PTHrp,and salts thereof, in which amino acid residues (22-31) form anamphipathic α-helix, said residues (22-31) selected from hydrophilicamino acids (Haa) and lipophilic amino acids (Laa) ordered in thesequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

In another aspect this invention provides DNA sequences, vectors, andplasmids for the recombinant synthesis of polypeptide analogs of PTH,PTHrp, and of the physiologically active truncated homologs and analogsof PTH and PTHrp, and salts thereof, in which amino acid residues(22-31) form an amphipathic s-helix, the sequence of said residues(22-31) selected from: (SEQ ID NOS: 26, 27, 28, 29, and 30).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the DNA sequence and enzyme restriction sites of asynthetic gene coding for a PTHrp(1-34) analog of this invention.

FIG. 2 outlines the preparation of a plasmid incorporating a PTHrp(1-34)analog gene.

FIG. 3 outlines the preparation of a plasmid incorporating two copies ofa PTHrp(1-34) analog gene.

FIG. 4 outlines the preparation of a plasmid incorporating four copiesof a PTHrp(1-34) analog gene.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions

The one- and three-letter abbreviations for the various commonnucleotide bases and amino acids are as recommended in Pure Appl. Chem.31, 639-645 (1972) and 40, 277-290 (1974) and comply with 37 CFR §1.822(55 FR 18245, May 1, 1990). The abbreviations represent L-amino acidsunless otherwise designated as D- or D,L-. Certain amino acids, bothnatural and non-natural, are achiral, e.g. glycine. All peptidesequences are presented with the N-terminal amino acid on the left andthe C-terminal amino acid on the right.

Further abbreviations for other amino acids and compounds used hereinare:

    ______________________________________                                        hSer       homoserine                                                         hSerlac    homoserine lactone                                                 Nle        norleucine                                                         PEG2       radical of diethylene glycol methyl                                           ether, a.k.a. methoxydi(ethyleneoxy),                                         CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.2--, (MW = 119)                PEG5000    radical of poly(ethylene glycol methyl                                        ether), a.k.a. methoxypoly(ethyleneoxy),                                      CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.110--, (avg. MW = 5000)        PEGX       radical of poly(ethylene glycol methyl                                        ether), CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.n--, n = 2-225,                   (avg. MW = 100 to 10,000)                                          ______________________________________                                    

"Hydrophilic amino acid (Haa)" refers to an amino acid having at leastone hydrophilic functional group in addition to those required forpeptide bond formation, such as arginine, asparagine, aspartic acid,glutamic acid, glutamine, histidine, lysine, serine, threonine, andtheir homologs.

"Lipophilic amino acid (Laa)" refers to an uncharged, aliphatic oraromatic amino acid, such as isoleucine, leucine, methionine,phenylalanine, tryptophan, tyrosine, valine, and their homologs.

For the purposes of this invention, alanine is classified as"amphiphilic" i.e., capable of acting as either hydrophilic orlipophilic.

"Physiologically active truncated homolog or analog of PTH or PTHrp"refers to a polypeptide having a sequence comprising less than the fullcomplement of amino acids found in PTH or PTHrp which, however, elicitsa similar physiological response. The truncated PTH or PTHrp need not befully homologous with PTH or PTHrp to elicit a similar physiologicalresponse. PTH(1-34) and PTHrp(1-34) are preferred, but not exclusive,representatives of this group.

"Amphipathic α-helix" refers to the secondary structure exhibited bycertain polypeptides in which the amino acids assume an α-helicalconfiguration having opposing polar and nonpolar faces oriented alongthe long axis of the helix. The possibility of α-helical structure inthe polypeptide of interest may be explored to some extent by theconstruction of a "Schiffer-Edmundson wheel" (M. Schiffer and A. B.Edmundson, Biophys. J. 7, 121 (1967), incorporated herein by reference),of the appropriate pitch and noting the segregation of the hydrophilicand lipophilic residues on opposite faces of the cylinder circumscribingthe helix. Alternatively, empirical evidence, such as circular dichroismor x-ray diffraction data, may be available indicating the presence ofan α-helical region in a given polypeptide. An ideal α-helix has 3.6amino acid residues per turn with adjacent side chains separated by 100°of arc.

Eisenberg et al. in Nature 299: 371-374 (1982) and Proc. Nat. Acad. Sci.USA 81: 140-144 (1984) have combined a hydrophobicity scale with thehelical wheel to quantify the concept of amphipathic helices. The meanhydrophobic moment is defined as the vector sum of the hydrophobicitiesof the component amino acids making up the helix. The followinghydrophobicities for the amino acids are those reported by Eisenberg(1984) as the "consensus" scale: Ile 0.73; Phe 0.61; Val 0.54; Leu 0.53;Trp 0.37; Met 0.26 Ala 0.25; Gly 0.16; Cys 0.04; Tyr 0.02; Pro -0.07;Thr -0.18; Ser -0.26; His -0.40; Glu -0.62; Asn -0.64; Gln -0.69; Asp-0.72; Lys -1.10; Arg -1.76.

The hydrophobic moment, μ_(H), for an ideal α-helix having 3.6 residuesper turn (or a 100° arc (=360°/3.6) between side chains), may becalculated from:

    μ.sub.H =[(ΣH.sub.N sinδ(N-1).sup.2 +(ΣH.sub.N cosδ(N-1)).sup.2 ].sup.1/2,

where H_(N) is the hydrophobicity value of the N^(th) amino acid and thesums are taken over the N amino acids in the sequence with periodicityδ=100°. The hydrophobic moment may be expressed as the mean hydrophobicmoment per residue by dividing μ_(H) by N to obtain <μ_(H) >. A value of<μ_(H) > at 100°±20° of about 0.20 or greater is suggestive ofamphipathic helix formation. The <μ_(H) > values at 100° for hPTHrp(22-31) and hPTH (22-31) are 0.19 and 0.37, respectively.

Cornett, et al., in J. Mol. Biol., 195: 659-685 (1987) have furtherextended the study of amphiphathic α-helices by introducing the"amphipathic index" as a predictor of amphipathicity. They concludedthat approximately half of all known α-helices are amphipathic, and thatthe dominant frequency is 97 5° rather than 100°, with the number ofresidues per turn being closer to 3.7 than 3.6. While such refinementsare scientifically interesting, the basic approach of Eisenberg, et al.is sufficient to classify a given sequence as amphipathic, particularlywhen one is designing a sequence ab initio to form an amphipathicα-helix.

A substitute amphipathic e-helical amino acid sequence may lack homologywith the sequence of a given segment of a naturally occurringpolypeptide but elicits a similar secondary structure, i.e. an α-helixhaving opposing polar and nonpolar faces, in the physiologicalenvironment. Replacement of the naturally occurring amino acid sequencewith an alternative sequence may beneficially affect the physiologicalactivity, stability, or other properties of the altered parentpolypeptide. Guidance as to the design and selection of such sequencesis provided in J. L. Krstenansky, et al., FEBS Letters 242: 2, 409-413(1989), and J. P. Segrest, et al. Proteins: Structure, Function, andGenetics 8: 103-117 (1990) among others.

The ten amino acid amphipathic α-helix of this invention has theformula:

    Haa (Laa Laa Haa Haa).sub.2 Laa

wherein the Haa's are selected from the group of hydrophilic amino acidsand the Laa's are selected from the group of lipophilic amino acids, asdefined above. Assuming an idealized α-helix, residues 1, 4, 5, 8, and 9are distributed along one face (A) of the helix within about a 140° arcof each other, while residues 2, 3, 6, 7, and 10 occupy an opposing 140°arc on the other face (B) of the helix. Preferably, all the residues onone face are of the same polarity while all those on the other face areof the opposite polarity, i.e., if face A is all hydrophilic, face B isall lipophilic and vice versa. The skilled artisan will recognize thatwhile the helices of this invention are described by

    Haa(Laa Laa Haa Haa).sub.2 Laa,

the reverse sequence,

    Laa (Haa Haa Laa Laa).sub.2 Haa

will also meet the residue distribution criteria and is an equivalentdescriptor of the helices of this invention.

Alanine may be substituted for either hydrophilic or lipophilic aminoacids, since Ala can reside readily on either face of an amphipathicα-helix, although Ala₁₀ does not form an amphipathic α-helix. Generally,proline, cysteine, and tyrosine are not used; however, their presenceand other random errors in the sequence may be tolerated, e.g. ahydrophilic residue on the lipophilic face, as long as the remainingamino acids in the segment substantially conform to the hydrophilicface--lipophilic face division. A convenient method for determining if asequence is sufficiently amphipathic to be a sequence of this inventionis to calculate the mean hydrophobic moment, as defined above. If thepeak mean moment per residue at 100°±20° exceeds about 0.20, then thesequence will form an amphipathic helix and is a sequence of thisinvention.

For example, the mean hydrophobic moment per residue at 100° for (SEQ IDNO: 26), Xaa=Glu, is calculated as follows:

    ______________________________________                                        A.A.  H.sub.N δ (N-1)                                                                           H sin δ(N-1)                                                                     H cos δ(N-1)                           ______________________________________                                        E     -.62     0          0      -.62                                         L     .53     100       .52      -.17                                         L     .53     200       -.18     -.50                                         E     -.62    300       .34      -.31                                         K     -1.1    400       -.70     -.85                                         L     .53     500       .34      -.41                                         L     .53     600       -.46     -.27                                         E     -.62    700       .21      -.58                                         K     -1.1    800       -1.08    -.19                                         L     .53     900        0       -.53                                                                 Σ = 0.81                                                                         Σ = -4.43                              ______________________________________                                         μ.sub.H = [(0.81).sup.2 + (-4.43).sup.2 ]1/2 = 4.50                        < μ.sub.H > = 4.50/10 = 0.45                                          

For this sequence, the mean peak hydrophobic moment occurs at 92° andhas a value of 0.48.

In applying this concept to parathyroid hormone and parathyroid hormonerelated peptide, it was hypothesized that either or both regions (7-16)and (22-31) may exhibit α-helical secondary structure and could bereplaced with a non-homologous sequence having similar structuraltendencies, without loss of biological activity or induction ofimmunoreaction.

PREFERRED EMBODIMENTS

In one aspect this invention includes polypeptide analogs of thephysiologically active truncated homolog hPTHrp(1-34), as shown inFormula (I):

    Ala Val Ser Glu Xaa.sup.5 Gln Leu Leu His Asp Xaa.sup.11 Gly Xaa.sup.13 Ser Ile Gln Asp Leu Xaa.sup.19 Arg Xaa.sup.21 Xaa.sup.22-31 Xaa.sup.32 Xaa.sup.33 Xaa.sup.34 Term,

wherein: Xaa⁵ is His or Ala; Xaa¹¹ and Xaa¹³ are independently Lys, Arg,or Leu; Xaa¹⁹ and Xaa²¹ are independently Ala or Arg; Xaa²²⁻³¹ isselected from: ##STR4## Xaa³² is His or Lys; Xaa³³ is Thr, Glu, or Ala;Xaa³⁴ is Ala, hSer, Tyr, or Leu; and Term is Gly Arg Arg, lactone, OH orNR₂, where each R is H or (C_(1-C) ₄) alkyl; and their pharmaceuticallyacceptable salts. (Formula I)

A more specific aspect of the invention includes those polypeptides ofFormula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26), for which <μ_(H) > at100° exceeds 0.45. A still more specific aspect of the inventionincludes those Formula (I) polypeptides wherein Xaa²²⁻³¹ is (SEQ IDNO:26); Xaa¹¹ and Xaa¹³ are both Lys; and Xaa¹⁹ and Xaa²¹ are both Arg.Representative polypeptides include, but are not limited to: ##STR5##

Another aspect of this invention includes those polypeptides of Formula(I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26); Xaa¹¹ and Xaa¹³ are both Lys;and one of Xaa¹⁹ and Xaa²¹ is Arg and the other is Ala. Representativepolypeptides of this subgenus include, but are not limited to: ##STR6##

In another aspect this invention includes those polypeptides of Formula(I) wherein Xaa²²⁻³¹ is (SEQ ID NO: 26); one of Xaa¹¹ and Xaa¹³ is Leuand the other is Lys; and Xaa¹⁹ and Xaa²¹ are both Arg. Representativepolypeptides of this subgenus include, but are not limited to: ##STR7##

In another aspect this invention includes those polypeptides of Formula(I) wherein Xaa²²⁻³¹ is (SEQ ID NO:27), for which <μ_(H) > at 100°exceeds 0.50. A further aspect of this invention includes those Formula(I) polypeptides wherein Xaa²²⁻³¹ is (SEQ ID NO:27); Xaa¹¹ and Xaa¹³ areboth Lys or both Arg; and Xaa¹⁹ and Xaa²¹ are both Arg. Representativepolypeptides of this subgenus include, but are not limited to: ##STR8##

In another aspect this invention includes polypeptides of Formula (I)wherein Xaa²²⁻³¹ is (SEQ ID NO:28), for which <μ_(H) > at 100° is about0.25. Representative polypeptides of this subgenus include, but are notlimited to: ##STR9##

In another aspect this invention includes polypeptides of Formula (I)wherein Xaa²²⁻³¹ is (SEQ ID NO:29), for which <μ_(H) > at 100° is about0.28. Representative polypeptides of this subgenus include, but are notlimited to: ##STR10##

In another aspect this invention includes polypeptides of Formula (I)wherein Xaa²²⁻³¹ is (SEQ ID NO:30), for which <μ_(H) > at 100° is about0.29. Representative polypeptides of this subgenus include, but are notlimited to: ##STR11##

Still another aspect of this invention includes polypeptide analogs ofthe physiologically active truncated homolog bPTH(1-34), as shown inFormula (II):

    Xaa.sup.1 Val Ser Glu Ile Gln Xaa.sup.7 Xaa.sup.8 His Asn Leu Gly Lys His Leu Xaa.sup.16 Ser Xaa.sup.18 Xaa.sup.19 Arg Xaa.sup.21 Xaa.sup.22-31 His Asn Xaa.sup.34 Term,

wherein:

Xaa¹ is Ser or Ala;

Xaa⁷ is Leu or Phe;

Xaa⁸ is Met or Nle;

Xaa¹⁶ is Asn or Ser;

Xaa¹⁸ is Leu, Met, or Nle;

Xaa¹⁹ is Glu or Arg;

Xaa²¹ is Val or Arg;

Xaa²²⁻³¹ is selected from (SEQ ID N0:26, 27, 28, 29, and 30);

Xaa³⁴ is Phe or Tyr;

Term is OH or NR₂, where each R is H or (C₁ -C₄)alkyl; and thepharmaceutically acceptable salts thereof. (Formula II) Representativepolypeptides include, but are not limited to: ##STR12##

The skilled artisan will appreciate that numerous permutations of thepolypeptide analogs may be synthesized which will possess the desirableattributes of those described herein provided that an amino acidsequence having a mean hydrophobic moment per residue at 100°±20°greater than about 0.20 is inserted at positions (22-31).

Classical Synthesis of the Polypeptides

The polypeptides of the instant invention may be synthesized by methodssuch as those set forth in J. M. Stewart and J. D. Young, Solid PhasePeptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984)and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, AcademicPress, New York, (1973) for solid phase synthesis and E. Schroder and K.Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) forsolution synthesis. The disclosures of the foregoing treatises areincorporated by reference herein.

In general, these methods involve the sequential addition of protectedamino acids to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid and any reactive side chain groupare protected. This protected amino acid is then either attached to aninert solid support, or utilized in solution, and the next amino acid inthe sequence, also suitably protected, is added under conditionsamenable to formation of the amide linkage. After all the desired aminoacids have been linked in the proper sequence, protecting groups and anysolid support are removed to afford the crude polypeptide. Thepolypeptide is desalted and purified, preferably chromatographically, toyield the final product.

A preferred method of preparing the analogs of the physiologicallyactive truncated polypeptides, having fewer than about forty aminoacids, involves solid phase peptide synthesis. In this method theα-amino (N⁶⁰ ) functions and any reactive side chains are protected byacid- or base-sensitive groups. The protecting group should be stable tothe conditions of peptide linkage formation, while being readilyremovable without affecting the extant polypeptide chain. Suitableα-amino protecting groups include, but are not limited tot-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz),o-chlorobenzyloxycarbonyl, biphenylisopropyloxycarbonyl,t-amyloxycarbonyl (Amoc), isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxy-carbonyl, o-nitrophenylsulfenyl,2-cyano-t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and thelike, preferably t-butoxycarbonyl (Boc). Suitable side chain protectinggroups include, but are not limited to: acetyl, benzyl (Bzl),benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl,t-butyldimethylsilyl, 2-chlorobenzyl (Cl-z), 2,6-dichlorobenzyl,cyclohexyl, cyclopentyl, isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl(Tos), trimethylsilyl, and trityl.

In solid phase synthesis, the C-terminal amino acid is first attached toa suitable resin support. Suitable resin supports are those materialswhich are inert to the reagents and reaction conditions of the stepwisecondensation and deprotection reactions, as well as being insoluble inthe media used. Examples of commercially available resins includestyrene/divinylbenzene resins modified with a reactive group, e.g.,chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylatedco-poly-(styrene-divinylbenzene), and the like. Benzylated,hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred. Whenthe C-terminus of the compound is an amide, a preferred resin isp-methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin.

Attachment to the PAM resin may be accomplished by reaction of theN.sup.α -protected amino acid, preferably the Boc-amino acid, as itsammonium, cesium, triethylammonium, 1,5-diazabicyclo-[5.4.0]undec-5-ene,tetramethylammonium, or similar salt in ethanol, acetonitrile,N,N-dimethylformamide (DMF), and the like, preferably the cesium salt inDMF, with the resin at an elevated temperature, for example betweenabout 40° and 60° C., preferably about 50° C., for from about 12 to 72hours, preferably about 48 hours.

The N.sup.α -Boc-amino acid may be attached to the benzhydrylamine resinby means of, for example, an N,N'-diisopropylcarbodiimide(DIC)/1-hydroxybenzotriazole (HOBt) mediated coupling for from about 2to about 24 hours, preferably about 2 hours at a temperature of betweenabout 10° and 50° C., preferably 25° C. in a solvent such asdichloromethane or dimethylformamide, preferably dichloromethane.

The successive coupling of protected amino acids may be carried out bymethods well known in the art, typically in an automated peptidesynthesizer. Following neutralization with triethylamine or similarbase, each protected amino acid is preferably introduced inapproximately 1.5 to 2.5 fold molar excess and the coupling carried outin an inert, nonaqueous, polar solvent such as dichloromethane, DMF, ormixtures thereof, preferably in dichloromethane at ambient temperature.Representative coupling agents are N,N'-dicyclohexylcarbodiimide (DCC),N,N'-diisopropyl-carbodiimide (DIC) or other carbodiimide, either aloneor in the presence of 1-hydroxybenzotriazole (HOBt), O-acyl ureas,benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBop), N-hydroxysuccinimide, other N-hydroxyimides, or oximes.Alternatively, protected amino acid active esters (e.g. p-nitrophenyl,pentafluorophenyl and the like) or symmetrical anhydrides may be used.

At the end of the solid phase synthesis the fully protected peptide isremoved from the resin. When the linkage to the resin support is of thebenzyl ester type, cleavage may be effected by means of aminolysis withan alkylamine or fluoroalkylamine for peptides with an alkylamideC-terminus, or by aminolysis with, for example, ammonia/methanol orammonia/ethanol for peptides with an unsubstituted amide C-terminus, ata temperature between about -10° and 50° C., preferably about 25° C.,for between about 12 and 24 hours, preferably about 18 hours. Peptideswith a hydroxy C-terminus may be cleaved by HF or other strongly acidicdeprotection regimen or by saponification. Alternatively, the peptidemay be removed from the resin by transesterification, e.g., withmethanol, followed by aminolysis or saponification. The protectedpeptide may be purified by silica gel chromatography.

The side chain protecting groups may be removed from the peptide bytreating the aminolysis product with, for example, anhydrous liquidhydrogen fluoride in the presence of anisole or other carbonium ionscavenger, treatment with hydrogen fluoride/pyridine complex, treatmentwith tris(trifluoroacetyl)boron and trifluoroacetic acid, by reductionwith hydrogen and palladium on carbon or polyvinylpyrrolidone, or byreduction with sodium in liquid ammonia, preferably with liquid hydrogenfluoride and anisole at a temperature between about -10° and +10° C.,preferably at about 0° C., for between about 15 minutes and 2 hours,preferably about 1.5 hours.

For peptides on the benzhydrylamine resin, the resin cleavage anddeprotection steps may be combined in a single step utilizing liquidhydrogen fluoride and anisole as described above.

The solution may be desalted (e.g. with BioRad AG-3® anion exchangeresin) and the peptide purified by a sequence of chromatographic stepsemploying any or all of the following types: ion exchange on a weaklybasic resin in the acetate form; hydrophobic adsorption chromatographyon underivatized co-poly(styrene-divinylbenzene), e.g. Amberlite® XAD;silica gel adsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g. on Sephadex®G-25; counter-current distribution; or high performance liquidchromatography (HPLC), especially reversed-phase HPLC on octyl- oroctadecylsilylsilica (ODS) bonded phase column packing.

Thus, another aspect of the present invention relates to processes forpreparing polypeptides and pharmaceutically acceptable salts thereofwhich processes comprise sequentially condensing protected amino acidson a suitable resin support, removing the protecting groups and resinsupport, and purifying the product, to afford analogs of thephysiologically active truncated homologs and analogs of PTH and PTHrp,preferably of PTH(1-34) and PTHrp(1-34), in which the amino acids atpositions (22-31) form an amphipathic α-helical peptide sequence, asdefined above.

Recombinant Synthesis of the Polypeptides

Alternatively, the polypeptides of this invention may be prepared bycloning and expression of a gene encoding for the desired polypeptide.In this process, a plasmid containing the desired DNA sequence isprepared and inserted into an appropriate host microorganism, typicallya bacteria, such as E. coli, or a yeast, such as Saccharomycescerevisiae, inducing the host microorganism to produce multiple copiesof the plasmid, and so of the cDNA encoding for the polypeptide analogsof this invention.

First, a synthetic gene coding for the selected PTH or PTHrp analog isdesigned with convenient restriction enzyme cleavage sites to facilitatesubsequent alterations. Polymerase chain reaction (PCR), as taught byMullis in U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporated herein byreference, may be used to amplify the sequence.

The amplified synthetic gene may be isolated and ligated to a suitableplasmid, such as a Trp LE plasmid, into which four copies of the genemay be inserted in tandem. Preparation of Trp LE plasmids is describedin U.S. Pat. No. 4,738,921 and European Patent Publication No. 0212532,incorporated herein by reference. Trp LE plasmids generally produce 8-10times more protein than Trp E plasmids. The multi-copy gene may then beexpressed in an appropriate host, such as E. coli or S. cerevisiae.

The specific expression vector used herein was Trp LE 18 Prot (Ile³,Pro⁵) containing the following elements: a pBR322 fragment (EcoRI-BamHI)containing the ampicillin resistant gene and the plasmid origin ofreplication; an EcoRI-SacII fragment containing the trp promoter and thetrpE gene; an HIV protease (Ile³, Pro⁵) gene fragment (SacII-HindIII); abGRF gene fragment (HindIII-BamHI); and a transcription terminator fromE. coli rpoc gene. The HIV protease and bGRF gene fragments are notcritical and may be replaced with other coding sequences, if desired.

The expressed multimeric fusion proteins accumulate intracellularly intostable inclusion bodies and may be separated by centrifugation from therest of the cellular protein. The isolated fusion protein is convertedto the monomeric PTH or PTHrp analog and may be purified by cationexchange and/or reverse phase HPLC.

Alternative methods of cloning, amplification, expression, andpurification will be apparent to the skilled artisan. Representativemethods are disclosed in Maniatis, et al., Molecular Cloning, aLaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989),incorporated herein by reference.

Utility and Administration

The polypeptides of this invention are useful for the prevention andtreatment of a variety of mammalian conditions manifested by loss ofbone mass. In particular, the compounds of this invention are indicatedfor the prophylaxis and therapeutic treatment of osteoporosis andosteopenia in humans.

In general, the polypeptides of this invention, or salts thereof, areadministered in amounts between about 0.01 and 1 μg/kg body weight perday, preferably from about 0.07 to about 0.2 μg/kg body weight per day.For a 50 kg human female subject, the daily dose of active ingredient isfrom about 0.5 to about 50 μgs, preferably from about 3.5 to about 10μgs. In other mammals, such as horses, dogs, and cattle, higher dosesmay be required. This dosage may be delivered in a conventionalpharmaceutical composition by a single administration, by multipleapplications, or via controlled release, as needed to achieve the mosteffective results, preferably one or more times daily by injection.

The selection of the exact dose and composition and the most appropriatedelivery regimen will be influenced by, inter alia, the pharmacologicalproperties of the selected polypeptide, the nature and severity of thecondition being treated, and the physical condition and mental acuity ofthe recipient.

Representative delivery regimens include oral, parenteral (includingsubcutaneous, intramuscular and intravenous), rectal, buccal (includingsublingual), transdermal, and intranasal.

Pharmaceutically acceptable salts retain the desired biological activityof the parent polypeptide without toxic side effects. Examples of suchsalts are (a) acid addition salts formed with inorganic acids, forexample hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid and the like; and salts formed with organic acids suchas, for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acids, naphthalene disulfonicacids, polygalacturonic acid and the like; (b) base addition saltsformed with polyvalent metal cations such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and thelike; or with an organic cation formed from N,N'-dibenzylethylenediamineor ethylenediamine; or (c) combinations of (a) and (b), e.g., a zinctannate salt and the like.

A further aspect of the present invention relates to pharmaceuticalcompositions comprising as an active ingredient a polypeptide of thepresent invention, or pharmaceutically acceptable salt thereof, inadmixture with a pharmaceutically acceptable, non-toxic carrier. Asmentioned above, such compositions may be prepared for parenteral(subcutaneous, intramuscular or intravenous) administration,particularly in the form of liquid solutions or suspensions; for oral orbuccal administration, particularly in the form of tablets or capsules;for intranasal administration, particularly in the form of powders,nasal drops or aerosols; and for rectal or transdermal administration.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985), incorporated herein by reference. Formulations for parenteraladministration may contain as excipients sterile water or saline,alkylene glycols such as propylene glycol, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. For oral administration, the formulation can be enhancedby the addition of bile salts or acylcarnitines. Formulations for nasaladministration may be solid and may contain excipients, for example,lactose or dextran, or may be aqueous or oily solutions for use in theform of nasal drops or metered spray. For buccal administration typicalexcipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

When formulated for nasal administration, the absorption across thenasal mucous membrane may be enhanced by surfactant acids, such as forexample, glycocholic acid, cholic acid, taurocholic acid, ethocholicacid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid,glycodeoxycholic acid, cyclodextrins and the like in an amount in therange between about 0.2 and 15 weight percent, preferably between about0.5 and 4 weight percent, most preferably about 2 weight percent.

Delivery of the compounds of the present invention to the subject overprolonged periods of time, for example, for periods of one week to oneyear, may be accomplished by a single administration of a controlledrelease system containing sufficient active ingredient for the desiredrelease period. Various controlled release systems, such as monolithicor reservoir-type microcapsules, depot implants, osmotic pumps,vesicles, micelies, liposomes, transdermal patches, iontophoreticdevices and alternative injectable dosage forms may be utilized for thispurpose. Localization at the site to which delivery of the activeingredient is desired is an additional feature of some controlledrelease devices, which may prove beneficial in the treatment of certaindisorders.

One form of controlled release formulation contains the polypeptide orits salt dispersed or encapsulated in a slowly degrading, non-toxic,non-antigenic polymer such as copoly(lactic/glycolic) acid, as describedin the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. Pat. No.4,675,189, incorporated by reference herein. The compounds or,preferably, their relatively insoluble salts, may also be formulated incholesterol or other lipid matrix pellets, or silastomer matriximplants. Additional slow release, depot implant or injectableformulations will be apparent to the skilled artisan. See, for example,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinsoned., Marcel Dekker, Inc., New York, 1978, and R. W. Baker, ControlledRelease of Biologically Active Agents, John Wiley & Sons, New York,1987, incorporated by reference herein.

The following specific Examples are intended to illustrate the synthesisand testing of representative compounds of the invention and should notbe construed as limiting the scope of the claims. In the Examples,"m.p." is melting point, "[α]_(D) ²⁵ " is the optical activity at 25° C.at the given concentration in the indicated solvent, "FAB" is fast atombombardment mass spectrometry, and "AAA" is amino acid analysis, withexpected values in parentheses following the observed values. The aminoacid analyses were conducted on a Hewlett-Packard AminoQuant Analyzerfollowing the manufacturer's recommended protocols. Primary amino acidswere derivatized with o-phthalaldehyde; secondary amino acids with Fmoc.Fluorescent detection of the derivatized amino acids was used forquantification. The protected amino acids were obtained from AppliedBiosystems Inc. (Foster City, Calif.).

EXAMPLE I

Compound 1 (SEQ ID NO:5) was prepared on 0.5 mmol scale by the solidphase method on 4-methylbenzhydrylamine resin, using an automatedApplied Biosystems Model 430A peptide synthesizer. The α-amino groupswere protected with t-butoxycarbonyl (Boc). The side chain protectinggroups were: benzyl (Bzl) for Asp, Glu, and Ser; tosyl (Tos) for Arg;benzyloxymethyl (Bom) for His; and 2-chlorobenzyl (Cl-z) for Lys. Theamino acids were coupled sequentially usingN,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBt) followingStewart and Young (supra). After each amino acid coupling, the peptidewas acetylated using a mixture of acetic anhydride anddiisopropylethylamine in N-methylpyrrolidone. The completed peptide wascleaved from the resin with simultaneous deprotection of the side chainprotecting groups using anhydrous HF (25 mL) in the presence of anisole(2.5 mL) at -10° C. for 30 minutes and at 0° C. for 60 minutes. Afterevaporation of the HF in vacuo, the residue was washed with anhydrousether, and the crude peptide extracted with 10% acetic acid.Lyophilization of the 10% acetic acid extract gave 900 mgs of crudeproduct. The peptide was purified by medium pressure ODS reversed phasecolumn chromatography using a gradient of 22-45% CH₃ CN in 0.1% TFA. Theproduct eluted in three fractions, which were concentrated andlyophilized to give 130 mgs of white solid of >98% purity.

Compound 1

AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA-NH₂ (SEQ ID NO: 7)

Physical Data:

m.p. 150°-159° C. [α]_(D) ²⁵ -34.88° (c 0.16, H₂ O)

FAB (C₁₇₅ H₃₀₀ N₅₆ O₅₁): [M+H]⁺ 4005.5

AAA: Asp, 1.9(2); Glu, 5.6(6); Ser, 1.6(2); His, 2.7(3); Gly, 1.0(1);Thr, 0.9(1); Ala, 1.9(2); Arg, 2.8(3); Val, 1.0(1); Ile, 0.9(1); Leu,7.3(8); Lys, 4.0(4).

Similarly, Compounds 2 and 5-18 were prepared and characterized,substituting PAM resin as needed for the synthesis of hydroxy-terminalpolypeptides.

Compound 2

AVSEHOLLHDKGKSinDLRRRELLEKLLEKLHT -OH (SEQ ID NO: 6)

Physical Data:

m.p. 154°-170° C. [α]_(D) ²⁵ -49.35° (c 0.46, H₂ O)

FAB (C₁₇₅ H₃₀₁ N₅₇ O₅₀): [M+H]⁺ 4005.0

AAA: Asp, 2.1(2); GlU, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly 1.1(1);Thr, 1.0(1); Ala, 1.9(2); Arg, 3.0(3); Val, 1.2(1); Ile, 1.0(1); Leu,7.8(8); Lys, 4.2(4).

Compound 5

AVSEHQLLHDKGKSIQDLRRRELLERLLERLHT-OH (SEQ ID NO: 15)

Physical Data:

m.p. 147°-165° C. [α]_(D) ²⁵ -49.17° (c 0.66, H₂ O)

FAB (C₁₇₅ H₂₉₉ N₅₉ O₅₂): [M+H]⁺ 4061

AAA: Asp, 2.1(2); Gly, 6.1(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 5.0(5); Val, 1.0(1); Ile, 0.9(1); Leu,7.7(8); Lys, 1.9(2).

Compound 6

AVSEHOLLHDRGRSIODLRRRELLERLLERLHTA-OH (SEQ ID NO:16)

Physical Data:

m.p. 150°-170° C. [α]_(D) ²⁵ -48.65° (c 0.54, H₂ O)

FAB (C₁₇₅ H₂₉₉ N₆₃ O₅₂): [M+H]⁺ 4118.0

AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.2(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 6.9(7); Val, 1.0(1); Ile, 1.0(1); Leu,7.8(8); Lys, 1.9(2).

Compound 7

AVSEHQLLHDRGRSIODLRRRELLERLLKRLHTA-OH (SEQ ID NO: 17)

Physical Data:

m.p. 177°-182° C. [α]_(D) ²⁵ -46.17° (c 0.14, H₂ O)

FAB (C₁₇₆ H₃₀₄ N₆₄ O₅₀): [M+H]⁺ 4117

AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1);Thr, 0.9(1); Ala, 1.9(2); Arg, 6.7 (7); Val, 1.0(1); Ile, 1.0(1); Lys,7.7(8); Lys, 0.9 (1).

Compound 8

AVSEHQLLHDKGKSIDLRRRELLEKLLRKLHTA-OH (SEQ ID NO:5)

Physical Data:

m.p. 147°-165° C. [α]_(D) ²⁵ -49.17° (c 0.66, H₂ O)

FAB (C₁₇₆ H₃₀₅ N₅₉ O₄₉): [M+H]⁺ 4033.0

AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.8(2); His, 2.7(3); Gly, 1.1(1);Thr, 0.9(1); Ala, 2.0(2); Arg, 3.9(4); Val, 1.0(1); Ile, 1.0(1); Leu,7.9(8); Lys, 4.0(4).

Compound 9

AVSEHOLLHDKGKSIODLRRRELLEKLLEKLHTAGRR-OH (SEO ID NO: 10)

Physical Data:

m.p. 158°-160° C. [α]_(D) ²⁵ -44.76° (c 0.1, H₂ O)

FAB (C₁₈₉ H₃₂₆ N₆₄ O₅₅): [M]⁺ 4375.0

AAA: Asp, 2.0(2); Glu, 5.9or (6); Ser, 1.7 (2); His, 2.9(3); Gly,2.3(2); Thr, 1.0(1); Ala, 1.9(2); Arg, 5.0(5); Val, 1.2(1); Ile, 1.0(1);Leu, 7.8(8); Lys, 4.3(4).

Compound 10

AVSEAOLLHDLGKSIODLRRRELLEKLLKLHA-OH (SEQ ID NO:14)

Physical Data:

m.p. 170°-175° C. [α]_(D) ²⁵ -31.59° (c 0.54, H₂ O)

FAB (C₁₇₄ H₃₀₀ N₅₂ O₅₁): [M]₊ 3936.0

AAA: Asp, 2.0(2); Glu, 6.0(6); Ser, 1.8(2); His, 2.0(2); Gly, 1.2(1);Ala, 3.0(3); Arg, 2.8(3); Val, 1.1(1); Ile, 1.0(1); Leu, 9.9(10); Lys,3.0(3).

Compound 11

AVSEHOLLHDKGKSIODLRRRELLEKLLELLKEL-NH₂ (SEQ ID NO:11)

Physical Data:

m.p. 172°-174° C. [α]_(D) ²⁵ -43.29° (c 0.2, H₂ O)

FAB (C₁₇₉ H₃₁₁ N₅₅ O₅₂): [M]⁺ 4065.8

AAA: Asp, 2.2(2); Glu, 7.7(7); Ser, 1.7(2); His, 2.0(2); Gly, 1.0(1);Ala, 1.0(1); Arg, 3.0(3); Val, 1.1(1); Ile, 1.0(1); Leu, 9.3(9); Lys,5.1(5).

Compound 12

AVSEIOFXHNLGKHLSSXERVELLEKLEKLHNY-NH₂ (X=Nle, SEQ ID NO:23)

Physical Data:

m.p. 178° C. [α]_(D) ²⁵ -36.88° (c 0.4, H₂ O)

FAB (C₁₈₂ H₂₉₅ N₅₀ O₅₁): [M+H]⁺ 4001.6

AAA: Asp, 2.1(2); Glu, 6.5(6); Ser, 2.7(3); His, 3.1(3); Gly, 1.1(1);Ala, 1.0(1); Arg, 1.0(1); Tyr, 0.8(1); Val, 2.0(2); Phe, 1.0(1); Ile,0.9(1); Leu+Nle, 8.5(7+2); Lys, 3.1(3).

Compound 13

AVSEIOFXHNLGKHLSSXRRREKLHNY-NH₂ (X=Nle, SEQ ID NO: 24)

Physical Data:

m.p. 260° C. [α]_(D) ²⁵ -37.02° (c 0.2, H₂ O)

FAB (C₁₈₄ H₃₀₄ N₅₆ O₄₉): [M+H]⁺ 4084

AAA: Asp, 2.1(2); Glu, 5.5(5); Ser, 2.6(3); His, 3.1(3); Ala, 1.0(1);Gly, 1.1(1); Arg, 3.2(3); Tyr, 1.0(1); Val, 1.0(1); Phe, 1.0(1); Ile,1.0(1); Leu, 9.0(9); Lys, 3.0(3).

Compound 14

AVSEHOLLHDKGKSIQDLRRRALAEALAEALHTA-NH₂ (SEQ ID NO:20)

Physical Data:

m.p. 190°-225° C. [α]_(D) ²⁵ -56.58° (c 0.36, H₂ O)

FAB (C₁₆₁ H₂₇₂ N₅₄ O₄₉): [M+H]⁺ 3747.0

AAA: Asp, 2.1(2); Glu, 4.9(5); Ser, 1.7(2); His, 2.6(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 7.6(7); Arg, 2.8(3); Val, 1.2(1); Ile, 1.0(1); Leu,6.6(6); Lys, 1.9(2).

Compound 15

AVSEHOLLHDKGKSIODLARRELLEKLLEKLHTA-NH₂ (SEO ID NO:12)

Physical Data:

m.p. 170°-180° C. [α]_(D) ²⁵ -48.19° (c 0.2, H₂ O)

FAB (C₁₇₂ H₂₉₃ N₅₃ O₅₁): [M+H]⁺ 3919.0

AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.7(2); His, 3.1(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu,8.0(8); Lys, 4.4(4).

Compound 16

AVSEHOLLHDKGKSIODLRRAELLEKLLEKLHTA-NH₂ (SEO ID NO: 13)

Physical Data:

m.p. 190°-195° C. [α]_(D) ²⁵ -50.50° (c 0.4, H₂ O)

FAB (C₁₇₂ H₂₉₃ N₅₃ O₅₁): [M+H]⁺ 3919.0

AAA: Asp, 2.1(2); Glu, 6.0(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu,7.5(8); Lys, 4.2(4).

Compound 17

AVSEHOLLHDKGKSIQDLRRRSLLSSLLSSLHTA-NH₂ (SEQ ID NO: 21)

Physical Data:

m.p. 195°-204° C. [α]_(D) ²⁵ -67.11° (c 0.3, H₂ O)

FAB (C₁₆₃ H₂₈₀ N₅₄ O₅₀): [M+H]⁺ 3796.0

AAA: Asp, 2.1(2); Glu, 2.9(3); Ser, 6.8(7); His, 3.1(3); Gly, 1.2(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Val, 1.0(1); Ile, 1.0(1); Leu,8.2(8); Lys, 2.0(2).

Compound 18

AVSEHOLLHDKGKSIODLRRRAFYDKVAEKLHTA-NH₂ (SEQ ID NO: 22)

Physical Data:

m.p. 200°-207° C. [α]_(D) ²⁵ -60.26° (c 0.6, H₂ O)

FAB (C₁₇₄ H₂₈₄ N₅₆ O₅₀): [M+H]⁺ 3960.0

AAA: Asp, 2.9(3); Glu, 3.5(4); Ser, 1.4(2); His, 2.6(3); Gly, 0.9(1);Thr, 1.0(1); Ala, 4.0(4); Arg, 3.0(3); Tyr, 0.9(1); Val, 1.9(2); Phe,1.1(1); Ile, 0.9(1); Leu, 3.6(4); Lys, 4.1(4).

EXAMPLE II

[Met³⁴, Ala³⁵ ] Compound 1, AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTMA-NH₂,(SEQ ID NO:25), was prepared and purified following the procedures ofExample I. This polypeptide was converted to the homoserine lactone asfollows. The purified peptide (160 mgs) was dissolved in 44% formic acid(4 mL). This solution was combined with a premixed solution of cyanogenbromide (700 mgs) and phenol (1.6 mgs) in 44% formic acid (4 mL) at 0°C. The solution was stirred at 0° C. for 2 hr and at room temperaturefor 2 hrs. The formation of the product was monitored by HPLC (Vydac®C-18, 300 A, 4.6×250 mm, flow of 1.2 mL/min, gradient 25-45%acetonitrile in 0.1% TFA over 10 min). The reaction was complete within4 hr. Half of the sample was concentrated and purified by preparativeRP-HPLC (Vydac® C-18, gradient 25-45% acetonitrile in 0.1% TFA). Thehomoserine lactone peptide fractions were pooled and lyophilized to give28 mgs of white solid of >95% purity, Compound 4.

Compound 4

AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX (X=hSerlac, SEQ ID NO: 9)

Physical Data:

m.p. 138°-142° C. [α]_(D) ²⁵ -50.66° (c 0.1, H₂ O)

FAB (C₁₇₆ H₂₉₉ N₅₅ O₅₂): [M+H]⁺ 4017.61

AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.8(2); His, 3.0(3); Thi, 1.1(1);Ala, 1.1(1); Arg, 2.7(3); Val, 1.0(1); Ile, 1.0(1); Leu, 8.2(8); Lys,3.8(4); Gly 1.09(1); hSer, 1.09(1).

EXAMPLE III

To prepare the homoserine amide, the crude hSerlactone analog, Compound4, was concentrated and treated with 25 mL saturated NH₃ in methanol.The solution was stirred at 0° C. for 2 hr and at room temperature for16 hr. The reaction was monitored by HPLC (Vydac® C-18, 300 Å, 4.6×250mm, flow of 1.2 mL/min, gradient 20-45% acetonitrile in 0.1% TFA) andwas complete within 18 hr. The solution was concentrated and purified bypreparative RP-HPLC (Vydac® C-18, gradient of 25-45% acetonitrile in0.1% TFA). The homoserine amide peptide fractions were pooled andlyophilized to give 30 mgs of white solid of >98% purity, Compound 3.

Compound 3

AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX-NH₂ (X=hSer, SEQ ID NO:8)

Physical Data:

m.p. 138°-142° C. [α]_(D) ²⁵ -45.97° (c 0.25, H₂ O)

FAB (C₁₇₆ H₃₀₂ N₅₆ O₅₂): [M+H]⁺ 4033.9

AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.6(2); His, 2.8(3); Gly, 0.97(1);hSer, 0.97(1); Thi, 1.0(1); Ala, 1.0(1); Arg, 2.9(3); Val, 1.0(1); Ile,1.0(1); Leu, 7.6(8); Lys, 3.9(4).

EXAMPLE IV

Following Example I, the protected peptide-resinBocAVS(Bzl)E(OBz)H(Bom)QLLHD(OBzl)R(Ts)GR(Ts)S(Bzl)IQD(OBz)LR(Ts)R(Ts)E(OBz)LLE(OBzl)R(Ts)LLK(Fmoc)R(Ts)LH(Bom)T(Bzl)AO-PAM wassynthesized on a 0.35 mmol scale. All N.sup.α groups were protected witht-butoxycarbonyl (Boc); side chain protecting groups were as indicated.After completion of the synthesis, the peptide resin was treated with 50mL of 20% piperidine in dimethylformamide (DMF) at room temperature for30 minutes to remove the fluorenylmethoxycarbonyl (Fmoc) protectinggroup on lysine. The resin was washed successively with DMF, MeOH, CH₂Cl₂ and dried to give 1.6 g partially protected peptide. 0.8 g (0.175mmol) of the partially protected peptide was acylated on lysine with0.44 g (0.3 mmol) of methoxydi(ethyleneoxy) acetic acid [pEG(2)CH₂ COOH]in the presence of 0.16 g (0.3 mmol)benzotriazolyloxy-tris(pyrrolidino)phosphonium hexafluorophosphate(PyBop) and 0.067 g (0.525 mmol) of diisopropylethyl amine (DIEA) in 20mL DMF at room temperature for 5 hrs. After 5 hrs., the resin wasfiltered and washed successively with DMF, MeOH and CH₂ Cl₂. Theacylation step was repeated twice until negative ninhydrin result on theresin was obtained. The final peptide was cleaved from the resin withremoval of the side chain protecting groups and purification as inExample I; 100 mgs of Compound 19 were obtained. ##STR13##

Physical Data:

m.p. 145°-195° C. [α]_(D) ²⁵ -44.60° (c 0.2, H₂ O)

FAB (C₁₈₃ H₃₁₆ N₆₄ O₅₄): [M+H]⁺ 4276.2

AAA: Asp, 2.1(2); Glu, 5.0(5); Ser, 1.6(2); His, 2.9(3); Gly, 0.9(1);Thr, 1.9(2); Arg, 7.1(7); Val, 1.1(1); Ile, 1.0(1); Leu, 8.0(8); Lys,0.9(1).

EXAMPLE V

Peptide was synthesized, cleaved and purified in the same manner as inExample IV except 2-methoxypoly(ethyleneoxy) acetic acid [PEG(5000)CH₂CO₂ H] was used as the acylating agent. 0.8 g (0.175 mmol) of partiallyprotected peptide yielded 300 mgs of pure Compound 20. ##STR14##

Physical Data:

m.p. 105° C. [α]_(D) ²⁵ -22.95° (c 0.11, 50% aq. HOAc)

AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.6(2); His, 2.6(3); Gly, 1.1(1);Thr, 1.1(1); Arg, 7.3(7); Val, 0.8(1); Ile, 0.9(1); Leu, 8.3(8); Lys,1.1(1); Ala, 1.8(2).

EXAMPLE VI

Synthesis of hPTHrp(1-34) analog gene

A synthetic gene coding for the hPTHrp(1-34) analog Compound 4 (SEQ IDNO:9) was designed having the nucleotide sequence and enzyme restrictionsites shown in FIG. 1.

The requisite oligodeoxynucleotides were prepared with a DNA synthesizer(Milligen/Biosearch) using the phosphoramidite process of Sinha, et al.,Nucleic Acid Research 12, 4539-4557 (1984), incorporated herein byreference. After deprotection, the crude oligonucleotides were purifiedby gel electrophoresis on preparative 15% polyacrylamide gels. Theoligonucleotides were located with UV, excised from the gel, desaltedover Waters c18 Sep-pak® cartridges, and lyophilized.

Amplification via polymerase chain reaction (PCR) was carried out on aPerkin-Elmer Cetus thermal cycler, with 25 cycles of: 94° C. for 1minute, 50° C. for 2 minutes, and 72° C. for 3 minutes, using reagents,including Taq polymerase, from the "GeneAmp" DNA amplification kit(Perkin-Elmer Cetus).

Two overlapping oligonucleotides, an 88mer (2 μg), PTH3 (SEQ ID NO:31):##STR15## were prepared as the template DNA sequence for the hPTHrp(1-34) analog gene. Utilizing the two flanking primers, PTHPCRi:CCTCTAGATC TCCGCGGCGC TAG (SEQ ID NO:33) and PTHPCR2: CCTCGAAGCTTATGCATCAT TATC (SEQ ID N0:34), the entire gene was amplified by PCR.The amplified DNA products were purified by gel electrophoresis on 4%NuSieve® agarose gel. The band containing the synthetic hPTHrp(1-34)analog gene, approximately 150 bases in length, was excised from the geland approximately 200 ng of DNA isolated by Elu-Quik® glass gel DNAextraction (Schleicher & Schuell, Keene, N.H.).

EXAMPLE VII

Molecular Cloning of an hPTHrp(1-34)1 Analog Gene

To subclone the hPTHrp(1-34) analog gene of Example VI, 200 ng of theamplified DNA was isolated and cut by restriction enzymes Hind III andSac II. As shown in FIG. 2, the DNA was ligated to 2 μg TrpLE 18 Prot(Ile³, Pro⁵) plasmid previously cleaved with Hind III and Sac II.

The resulting plasmid, TrpLE 18 hPTHrp(1-34)1, containing one copy ofthe hPTHrp(1-34) analog gene was then transformed into competent E. coliHB 101 cells (CLONTECH, Palo Alto, Calif.). Transformants were subjectedto PCR analysis to verify insertion. Transformed cell colonies wereselected and boiled in 200 μL of water for 5 minutes; 2 μL weresubjected to PCR with two primers flanking the insert. The PCR productwas then analyzed on 1% agarose gel to confirm the presence of one copyof the hPTHrp(1-34) gene insert. TrpLE 18 hPTHrp(1-34)1 construct wasthen verified by DNA sequencing on an automated DNA sequencer (AppliedBiosystems Model 373A, Foster City, Calif.) using the vendor's Dye DeoxyTerminator Sequencing kit.

EXAMPLE VIII

Construction of a Trp LE 18 vector containing multiple copies of thehPTHrp(1-34) analog gene

Unique Nhe I and Xba I restriction sites are located near the beginningand end of the hPTHrp(1-34) analog gene sequence. These two sites, whichrecognize different sequences, but produce identical single strandcohesive termini, allow the construction of multiple copy hPTHrp (1-34)genes within the Trp LE 18 vector.

The strategy for constructing repeated hPTHrp(1-34) sequences in tandemis outlined in FIG. 3. In separate reactions, 5 μg of plasmid Trp LE 18hPTHrp(1-34)1 containing a single copy of the gene was cleaved with BamHI+Nhe I and Xba I+Bam HI. From each digest, about 300 ng of thefragment containing the hPTHrp(1-34) analog gene was isolated. These twofragments were mixed and ligated to form the Trp LE 18 hPTHrp(1-34)2plasmid. This plasmid was used to transform competent E. coli HB 101cells. Sizing of the transformed PCR products on 1% agarose gel was usedto determine the presence of two copies of the hPTHrp(1-34) gene insert.TrpLE 18 hPTHrp(1-34)2 containing two copies of the gene was thenconfirmed by DNA sequencing. The correct fusion of the two hPTHrp(1-34)genes results in the elimination of Nhe I and Xba I sites at thejunction. This makes the remaining Xba I and Nhe I sites flanking thetandem genes unique.

By repeating this process the final plasmid Trp LE 18 hPTHrp(1-34)4containing four copies of the hPTHrp(1-34) gene was constructed, asshown in FIG. 4. The sequence of Trp LE 18 hPTHrp(1-34)4 was found to becorrect by DNA sequence analysis.

EXAMPLE IX

Expression and Purification of TrP LE 18 hPTHrp(1-34)4

Induction of the Trp LE 18 hPTHrp(1-34)4. A starter culture of 50 mL ofLB culture media, J. H. Miller, "Experiments in Molecular Genetics,"p.431 (1972), incorporated herein by reference, containing 50 μg/mLampicillin and 100 μg/mL tryptophan, was inoculated with E. coli cellscontaining Trp LE 18 hPTHrp(1-34)4 plasmid, and grown overnight at 37°C. with vigorous shaking to an A₅₅₀ of about 6. Two liters of LB culturemedia for production were pre-warmed to 37° C. and seeded with 20 mL ofthe starter culture to give an A₅₅₀ of about 0.06. The culture was thengrown with vigorous shaking to an A₅₅₀ of between 0.6 and 0.8, whereupon2 mL of a 10 mg/mL solution of indole acrylic acid (IAA) was added.Growth was continued with good aeration for about 16 hr to a final A₅₅₀of about 6 (typically, between 4 and 10). The cells were concentrated bycentrifugation and resuspended in 500 mL of 50 mM Tris-HCl, pH 7.5, 0.1mM EDTA buffer solution (Tris buffer).

The suspension was sonicated using a Heat Systems-Ultrasonics, Inc.model 220F sonicator (equipped with a 3/4" horn) operated at 50% of fullcapacity to avoid overheating.

To determine the extent of induction, the whole cells were analyzed bySDS-PAGE. The gene products derived from the TrpLE 18 hPTHrp(1-34)4construct were seen as a major band of the predicted MW of approximately17,000. This accounts for as much as 10% of the total cellular protein.

Isolation of the Fusion Protein. The cell lysate was centrifuged for 15min. at about 3600×g to pellet the Trp LE 18 hPTHrp(1-34)4 fusionprotein; the supernatant was discarded. The pellet was resuspended in200 mL Tris buffer. (typically 40-80 A_(550/) mL).

EXAMPLE X

Processing of the Fusion Protein and Purification of homo-SerlactonehPTHrp(1-34) peptide

Cleavage of the methionine residues flanking the hPTHrp(1-34) multimericfusion protein with CNBr releases the desired homo-SerlactonehPTHrp(1-34) polypeptide, which was purified as described below.

CNBr Treatment of Fusion Protein. The washed pellet of TrpLE 18hPTHrp(1-34)4 fusion protein was resuspended by gently stirring in 60 mLof 70% formic acid (about 20 mg/mL total protein; typically materialfrom 1000 A₅₅₀ units of cells is dissolved in 3 mL). A few drops ofoctanol were added and N₂ bubbled through the solution for 20 minutesbefore adding 5.5 g CNBr. This reaction was allowed to proceed for 6hours at 25° C. before an equal volume of 50:50 MeOH:H₂ O was mixed withthe sample and subsequently removed by rotary evaporation. After 2 to 4repetitions of this process, the bulk of the formic acid and CNBr wereessentially removed. The sample was then evaporated to dryness,redissolved in 200 mL water and lyophilized for storage.

Purification of homo-Serlactone hPTHrp(1-34). The CNBr cleavedsupernatant was dialyzed against 50 mM KH₂ PO₄ pH 6.5 for 24 hours withmultiple changes. During dialysis, pH was maintained at 6.5. Afterdialysis, the precipitates were removed by high speed centrifugation.The supernatant was clarified through a Gelman 0.45μ filter device(Acrodisc 4184).

Cation Exchange Chromatography. Initial purification was accomplished bycation exchange chromatography on a Bio-Gel TSK-SP-5PW HPLC column(21.5×150 mm). Chromatographic conditions for a flow rate of 8 mL/minand a yield of approximately 12 mg of highly purified homo-SerlactonehPTHrp(1-34) peptide were:

1. Column equilibration in 50 mM KH₂ PO₄, pH 6.5.

2. Load 10 mL clarified supernatant (approximately 1.5 L culture brothor 2.4 g inclusion).

3. Wash column with 50 mM KH₂ PO₄ pH 6.5 containing 50 mM NaCl untilbaseline is stabilized.

4. Elute column with 50 mM KH₂ PO₄ pH 6.5 containing 90 mM NaCl. Collectfractions for about 45 minutes.

5. Analyze the 90 mM NaCl fractions for homo-Serlactone hPTHrp(1-34)content by C18 HPLC before pooling and storage.

Reverse Phase HPLC Chromatography. A reverse phase Poros R/H 4.6×100 mmcolumn (Perseptive Biosystems, Cambridge, Mass.) was used for the finalpurification step. The chromatographic conditions were as follows:

    ______________________________________                                        Mobile phase                                                                           A:     0.1% trifluoroacetic acid (TFA)/water                                  B:     0.1% trifluoroacetic acid (TFA)/CH.sub.3 CN                   TIME           FLOW     % B                                                   ______________________________________                                          0 min        4 ml/min 15                                                    5.0 min        4 ml/min 40                                                    5.2 min        4 ml/min 100                                                   6.8 min        4 ml/min 100                                                   7.0 min        4 ml/min 15                                                    ______________________________________                                    

Retention time of the homo-Serlactone hPTHrp(1-34), Compound 4, wasapproximately 2.943 minutes. The purified peptide was approximately 98%pure as determined by mass spectroscopy.

EXAMPLE XI

The compounds of this invention were evaluated for their effect on bonemass in ovariectomized rats, generally in accord with the procedures ofGunness-Hey and Hock, Metab. Bone Dis. 5: 177 181 (1984), incorporatedby reference herein.

Adult Sprague-Dawley female rats were acclimatized, weight grouped (n=9,10 or 12), and subjected to bilateral ovariectomy (OVX) or sham surgery.Dosing was initiated 17 days after surgery and continued for 20 days.Test compound was administered subcutaneously once a day in 2% ratserum/saline vehicle.

After 20 days of dosing, the rats were sacrificed and the right femursexcised. The femurs were cut in half and the distal half femurs (DHF)were further separated into trabecular bone (TB) and cortical bone (CB)by drilling out the trabeculae. Calcium was extracted and measured byCalcette calcium analyzer and expressed as mean bone Ca in mg/DHF/100 gbody weight.

The two sample t-test was used to compare OVX and sham groups. One-wayANOVA was used to compare OVX groups, followed by Fisher's LSD multiplecomparison to compare each treatment group to vehicle.

Ovariectomy induced substantial total bone loss, primarily fromtrabecular bone. Total bone calcium was 47 to 54% lower than forsham-operated controls.

bPTH(1-34) and hPTHrp(1-34) at 80 μg/kg/day provided statisticallysignificant increases in total bone calcium for treated OVX rats,ranging from 18 to 53%; however, there was no significant increase incortical bone calcium.

Compounds of this invention, dosed at 80 μg/kg/day, increased total bonecalcium by from 66 to 138% and trabecular calcium by from 87 to 128%.Cortical bone calcium, trabecular thickness, and bone volume were alsosignificantly increased over untreated OVX controls.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 34                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsn                              151015                                                                        SerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspValHis                              202530                                                                        AsnPhe                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaValSerGluIleGlnPheMetHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspValHis                              202530                                                                        AsnPhe                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerLeuGluArgValGluTrpLeuArgLysLysLeuGlnAspValHis                              202530                                                                        AsnPhe                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgPhePheLeuHisHisLeuIleAlaGluIleHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuArgLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note= "Xaa34 = homoserine"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrXaa                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note= "Xaa34 = homoserine lactone"                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrXaa                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAlaGlyArgArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuLys                              202530                                                                        GluLeu                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuAlaArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgAlaGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AlaValSerGluAlaGlnLeuLeuHisAspLeuGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        AlaLeu                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuLysArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 29                                                              (D) OTHER INFORMATION: /note= "Xaa29 =                                        lysine- (OCCH.sub.2 (OCH.sub.2 CH.sub.2).sub.2 OCH.sub.3)"                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuXaaArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 29                                                              (D) OTHER INFORMATION: /note= "Xaa29 =                                        lysine- (OCCH.sub.2 (OCH.sub.2 CH.sub.2).sub.110 OCH.sub.3 "                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuXaaArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgAlaLeuAlaGluAlaLeuAlaGluAlaLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgSerLeuLeuSerSerLeuLeuSerSerLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgAlaPheTyrAspLysValAlaGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa8 = norleucine"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /note= "Xaa18 = norleucine"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      AlaValSerGluIleGlnPheXaaHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerXaaGluArgValGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        AsnTyr                                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa8 = norleucine"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /note= "Xaa18 = norleucine"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AlaValSerGluIleGlnPheXaaHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerXaaArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        AsnTyr                                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrMetAla                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa8 = glutamic acid or                        arginine"                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note= "Sequence 26 is embedded at                     positions 22 to 31 of sequences 5, 6, 7, 8, 9, 10,                            11, 12, 13, and 14."                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GluLeuLeuGluLysLeuLeuXaaLysLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "Xaa8 = glutamic acid,                          lysine, or lysine-(OCCH2PEGX)"                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note= "Sequence 27 is embedded at                     positions 22 to 31 of sequences 15, 16, 17, 18,                               and 19. "                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GluLeuLeuGluArgLeuLeuXaaArgLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note= "Sequence 28 is embedded at                     positions 22 to 31 of sequence 20."                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      AlaLeuAlaGluAlaLeuAlaGluAlaLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note= "Sequence 29 is embedded at                     positions 22 to 31 of sequence 21."                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      SerLeuLeuSerSerLeuLeuSerSerLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note= "Sequence 30 is embedded at                     positions 22 to 31 of sequence 22."                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      AlaPheTyrAspLysValAlaGluLysLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 88 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CCTCTAGATCTCCGCGGCGCTAGCATGGCTGTTTCTGAACATCAGCTGCTTCATGACAAA60                GGTAAATCGATTCAAGATCTGAGACGTC88                                                (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CCTCGAAGCTTATGCATCATTATCTAGACATAGTATGCAGCTTTTCAAGCAGTTTCTCCA60                GCAGCTCGCGACGTCTCAGATCTTGAATCG90                                              (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CCTCTAGATCTCCGCGCGCTAGC23                                                     (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CCTCGAAGCTTATGCATCATTATC24                                                    __________________________________________________________________________

We claim:
 1. A modified parathyroid honnone related polypeptide (PTHrP),of the formula:Ala Val Ser Glu Xaa⁵ Gln Leu Leu His Asp Xaa¹¹ Gly Xaa¹³Ser Ile Gln Asp Leu Xaa¹⁹ Arg Xaa²¹ Xaa²²⁻³¹ Xaa³² Xaa³³ Xaa³⁴ Term,wherein: Xaa⁵ is His or Ala; Xaa¹¹ and Xaa¹³ are independently Lys, Arg,or Leu; Xaa¹⁹ and Xaa²¹ are independently Ala or Arg; Xaa²²⁻³¹ isselected from the group consisting of (SEQ ID NOS: 26, 27, 28, 29, and30); Xaa³² is His or Lys; Xaa³³ is Thr, Glu, or Ala; Xaa³⁴ is Ala, hSer,Tyr, or Leu; Term is OH, NR₂, lactone, or Gly Arg Arg, where R is H or(C₁ -C₄) alkyl; and the pharmaceutically acceptable salts thereof.
 2. Apolypeptide of claim 1 wherein Xaa²²⁻³¹ is (SEQ ID NO:26).
 3. Apolypeptide of claim 2 wherein Xaa¹¹ and Xaa¹³ are both Lys; and Xaa¹⁹and Xaa²¹ are both Arg.
 4. The polypeptide of claim 3 which is:##STR16##
 5. The polypeptide of claim 3 which is: ##STR17##
 6. Thepolypeptide of claim 3 which is: ##STR18##
 7. The polypeptide of claim 3which is: ##STR19##
 8. The polypeptide of claim 3 which is: ##STR20## 9.The polypeptide of claim 3 which is: ##STR21##
 10. The polypeptide ofclaim 3 which is: ##STR22##
 11. A polypeptide of claim 2 wherein Xaa¹¹and Xaa¹³ are both Lys; and one of Xaa¹⁹ and Xaa²¹ is Arg and the otheris Ala.
 12. The polypeptide of claim 11 which is: ##STR23##
 13. Thepolypeptide of claim 11 which is: ##STR24##
 14. A polypeptide of claim 2wherein one of Xaa¹¹ and Xaa¹³ is Leu and the other is Lys; and Xaa¹⁹and Xaa²¹ are both Arg.
 15. The polypeptide of claim 14 which is:##STR25##
 16. A polypeptide of claim 1 wherein Xaa²²⁻³¹ is (SEQ IDNO:27).
 17. A polypeptide of claim 16 wherein Xaa¹¹ and Xaa¹³ are bothLys or both Arg; and Xaa¹⁹ and Xaa²¹ are both Arg.
 18. The polypeptideof claim 17 which is: ##STR26##
 19. The polypeptide of claim 17 whichis: ##STR27##
 20. The polypeptide of claim 17 which is: ##STR28## 21.The polypeptide of claim 17 which is: ##STR29##
 22. The polypeptide ofclaim 17 which is: ##STR30##
 23. A polypeptide of claim 1 whereinXaa²²⁻³¹ is (SEQ ID NO:28).
 24. The polypeptide of claim 23 which is:##STR31##
 25. A polypeptide of claim 1 wherein Xaa²²⁻³¹ is (SEQ IDNO:29).
 26. The polypeptide of claim 25 which is: ##STR32##
 27. Apolypeptide of claim 1 wherein Xaa²²⁻³¹ is (SEQ ID NO:30).
 28. Apolypeptide of claim 27 which is: ##STR33##
 29. A polypeptide of claim 1produced by a process of solid phase synthesis, which process comprisessequentially coupling protected amino acids on a suitable resin support,removing the side chain and N.sup.α protecting groups, and cleaving thepolypeptide from the resin support.
 30. A polypeptide of claim 1produced by expressing a gene encoding said polypeptide.
 31. A modifiedparathyroid hormone (PTH) polypeptide, of the formula:Xaa¹ Val Ser GluIle Gln Xaa⁷ Xaa⁸ His Asn Leu Gly Lys His Leu Xaa¹⁶ Ser Xaa¹⁸ Xaa¹⁹ ArgXaa²¹ Xaa²²⁻³¹ His Asn Xaa³⁴ Term, wherein: Xaa¹ is Ser or Ala; Xaa⁷ isLeu or Phe; Xaa⁸ is Met or Nle; Xaa¹⁶ is Asn or Ser; Xaa¹⁸ is Leu, Met,or Nle; Xaa¹⁹ is Glu or Arg; Xaa²¹ is Val or Arg; Xaa²²⁻³¹ is selectedfrom the group consisting of (SEQ ID NOS: 26, 27, 28, 29, and 30); Xaa³⁴is Phe or Tyr; Term is OH or NR₂, where R is H or (C₁ -C₄) alkyl; andthe pharmaceutically acceptable salts thereof.
 32. A pharmaceuticalcomposition for the prevention or treatment of conditions characterizedby decreases in bone mass comprising an effective bone mass increasingamount of a polypeptide of claim 31, or salt thereof, and apharmaceutically acceptable carrier.
 33. A pharmaceutical composition inunit dosage form, for treating conditions characterized by decreases inbone mass, comprising from about 0.5 μg to about 50 μg of a polypeptideof claim 31, or salt thereof, and a pharmaceutically acceptable carrier.34. The polypeptide of claim 1 which is: ##STR34##
 35. The polypeptideof claim 31 which is: ##STR35##
 36. A polypeptide of claim 31 producedby a process of solid phase synthesis, which process comprisessequentially coupling protected amino acids on a suitable resin support,removing the side chain, and N.sup.α protecting groups, and cleaving thepolypeptide from the resin support.
 37. A polypeptide of claim 31produced by expressing a gene encoding said polypeptide.